Dissociated cell cultures of dopaminergic neurons from embryonic rat mesencephalon were grown in completely defined serum-free medium. Survival and growth of the neurons was assessed by quantifying the level of the dopamine uptake system. As part of a study on the role of soluble growth factors during this period, it was observed that replacement of medium with fresh medium led to the complete destruction of the cultures. This damage was blocked in the presence of an antagonist (APV) of the NMDA-type of glutamate receptors. An antagonist of non-NMDA receptors (CNQX) was ineffective. Excitatory amino acids (EAA) operating via NMDA receptors are responsible for culture damage. While microM levels of glutamate or glutamate agonists can damage the cultures, the fresh medium nominally contains no glutamate and amino-aid analysis of the medium after damage to cultures does not show the presence of glutamate. However, subsequent analysis of the medium with a more sensitive method demonstrated the presence of 8.0-9.0 microM glutamate in the medium. The source of this glutamate was determined to be the large amount of glutamine (2.0-4.0 mM) normally present in the medium. Surprisingly, subsequent use of medium containing "glutamate-free" glutamine still caused damage to cultures that was blocked by antagonists of the NMDA receptor. The ligand responsible of this damage and the mechanisms by which it operates are not currently known.